Permeability of various drugs across the blood-brain barrier (BBB) is significantly dependent on the expression and functional activity of specific efflux transporters located in the membrane of brain capillary endothelial cells (BCEC). Selective transient downregulation of the transporters will lead to the application of more effective and less toxic doses of therapeutic drugs against brain tumors or viral infections in CNS. Previously, antisense inhibitors have been shown to temporarily arrest the synthesis of major multidrug resistance agent, membrane P-glycoprotein, and promote reversal of the resistant cell phenotype. The more effective RNA interference mechanism has been recently discovered for selective switching off expression of various genes. Short hairpin RNA (siRNA) could be introduced into target cells through a plasmid DNA precursor using methods of non-viral gene therapy. However, targeted delivery of the pDNA to the cells of the BBB requires a good systemic carrier and selective vectors that bind to the BCEC. As such a carrier, polymer crosslinked Nanogel particles modified with the brain-specific homing peptides (BSHP) have been chosen for tranfection of the BBB by shRNA-encoding plasmid DNA with an ultimate goal suppressing the specific membrane proteins, drug efflux transporters, in the BBB. Specific BSHPs to be attached to the surface of the Nanogel and target delivery to the BBB have been selected in vivo from a vast amount of peptides in the phage display library. Nanogel is non-toxic and highly effective as a transfection agent in many cell lines and evidently, one of the carriers with great potential for systemic administration. The vectorized RNA Interference-Producing system (RIP system) could be used for bioengineering of the BBB permeability for therapeutic agents whose brain accessibility was hampered by specific drug efflux transporters. The central hypothesis of the proposal is that transient suppression of selected drug efflux transporters in the BBB via systemic transfection of brain endothelium using targeted RNAi-producing systems can result in significant enhancement of drug transport to the brain during chemotherapy of the CNS-related diseases. Our Specific aim 1 is to develop the BCEC-targeted Nanogel carriers for systemic delivery of plasmid DNA to the BBB. Specific aim 2 is the enhance transfection efficacy of the BCEC-targeted Nanogel carriers in vitro and in vivo. Specific aim 3 is to suppress selected drug efflux transporters in the BBB in vivo and temporary increase drug transport into the brain. In this Aim brain transport of several representive nucleoside analogue drugs will be assessed in animal model following the transient downregulation of drug efflux transporters in the BBB by the Nanogel-based RNAi-producing systems. [unreadable] [unreadable]